Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, and disease-associated mutations (1-4). However, it has recently been demonstrated that GPCRs do not only signal in this simplistic fashion, but rather activate a network of downstream effects comprised of parallel signal transduction pathways. GPCR ligands biased towards induction or blockade of specific signaling pathways may have different physiology compared with unbiased molecules, through selective engagement of a desired subset of signal cascades. For example, the melanocortin 4 receptor (MC4R) transduces its signal via coupling to Gs and adenylyl cyclase activation, and is involved in the regulation of energy homeostasis and chronic disease-associated cachexia. Recent studies indicate that classical antagonists do not mimic MC4R regulation by its endogenous ligand, Agouti-Related Protein (AgRP). Indeed, AgRP has several actions including antagonizing Gs-mediated adenylyl cyclase activation, inducing beta-arrestin recruitment and MC4R endocytosis (5), as well as stimulating Gi-mediated inhibition of adenylyl cyclase (6). As a result, the identification of small molecules that act as biased MC4R ligands, by blocking Gs protein coupling but stimulating beta-arrestin functions and/or Gi protein coupling, may lead to a better understanding of this receptor and its role in metabolic/wasting diseases.

The purpose of this assay is to identify compounds that act as antagonists of Melanotan II generated cAMP signaling via MC4R. This assay employs the cAMP Perkin Elmer LANCE Ultra cAMP competitive immunoassay kit and cells that stably express the Gs-coupled human receptor MC4R. This receptor signals through adenylyl cyclase to trigger the cAMP cascade. Labeled anti-cAMP antibody and Europium-labeled cAMP (Eu-cAMP) are included for TRFRET-based detection of receptor activity. In this assay, cells are incubated with test compounds, labeled cAMP antibody and Eu-cAMP. Binding of the labeled cAMP antibody to Eu-cAMP causes energy transfer from the Eu-cAMP molecule to the labeled-antibody, increasing well FRET. Ligand-mediated MC4R activation stimulates endogenous cellular cAMP production by the cells, which competes with the labeled cAMP for binding to the labeled cAMP-antibody, thereby reducing FRET. As designed, compounds that act as antagonists will prevent receptor activation, reduce cellular cAMP production, reduce levels of cAMP available to compete with labeled Eu-cAMP, leading to increased interactions between Eu-cAMP and the labeled anti-cAMP antibody, and resulting in increased well FRET. Compounds are tested in singlicate at a nominal concentration of 6.5 uM.

Protocol Summary:

The MC4R cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham's F-12 Nutrient Media (F-12) and Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day of the assay, 1500 cells in 3 uL of cell media (HBSS, 25 mM HEPES and 0.1% BSA) were seeded into each well of 1536-well microtiter plates. Then, 26 nL of test compound in DMSO or DMSO alone was added to the appropriate wells and plates were incubated at room temperature for 30 minutes at room temperature. Next, 1 uL of the MC4R agonist, Melanotan II (Mel II), in assay buffer ("EC Challenge" consisting of HBSS, 25 mM HEPES, 100 uM Ro 20-1724 and 0.1% BSA), or assay buffer alone were dispensed to the appropriate wells. After incubation for 1 hour at room temperature, 1 uL of Eu-cAMP (prepared in lysis buffer according to the manufacturer's protocol) followed by 1 uL of labled-anti-cAMP (prepared in lysis buffer according to the manufacturer's protocol) was added to each well. Incubation was continued at room temperature for 1 hour before measurement of well FRET. After excitation at 340 nm (with 30 nm bandwidth), well fluorescence was monitored at 671 nm (with 4 nm bandwidth), using the Envision microplate reader (Perkin Elmer). For each well, fluorescence was calculated to normalize assay data, according to the following mathematical expression:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned 'Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.